Immunoassays to detect diseases or disease susceptibility traits

Abstract

Disclosed are immunoassay methods for the diagnosis/prognosis of diseases and disease susceptibility traits associated with gene mutations that cause protein truncation or allelic loss. The levels of one or more targeted wild-type proteins expressed by a subject gene or genes are immunologically quantitated in biological samples. Results indicating that a targeted wild-type protein is not present in an assayed sample, or that approximately 50% of the normal amount of such a wild-type protein is present in an assayed sample are considered to be positive for a mutation in one or both alleles of a subject gene, and correlated with the disease or the disease susceptibility trait associated with that mutation or mutations. Normal cells, particularly normal peripheral blood lymphocytes, are preferred biological samples.

1. A method of screening for hereditary breast cancer or a predisposition to hereditary breast cancer in an organism, said screening method comprising:
 (a) isolating a biological sample containing normal cells from said organism; 
 (b) immunologically quantitating the levels of full-length BRCA1 and full-length BRCA2 in said normal cells; 
 (c) calculating the ratio of the level of full-length BRCA1 in said normal cells, to the level of full-length BRCA2 in said normal cells; and 
 (d) determining whether the ratio calculated in step (c) reflects about a 50% decrease from a normal level of BRCA1 or BRCA2, wherein said normal level of BRCA1 or BRCA2 is determined in individuals unaffected by said hereditary breast cancer or predisposition to hereditary breast cancer; 
 (e) whereby if the ratio calculated in step (c) indicates that there is about a 50% decrease from said normal level of either full-length BRCA1 or full-length BRCA2 in said normal cells, that the subject organism has hereditary breast cancer or has a predisposition to hereditary breast cancer. 
 
     
     
       2. The method of  claim 1  wherein step (d) comprises comparing the ratio calculated in step (c) to the mean of ratios calculated from the levels of full-length BRCA1 and full-length BRCA2 proteins in comparable biological samples from organisms of the same taxonomic classification as the subject organism, wherein said organisms of the same taxonomic classification as the subject organism are unaffected by said hereditary breast cancer or predisposition to hereditary breast cancer. 
     
     
       3. The method of  claim 1  wherein said organism is a vertebrate. 
     
     
       4. The method of  claim 3  wherein said vertebrate is a mammal. 
     
     
       5. The method of  claim 4  wherein said mammal is a human. 
     
     
       6. The method of  claim 1  wherein said about 50% decrease from the normal level of full-length BRCA1 or full-length BRCA2 is the result of a mutation selected from the group consisting of truncating-causing mutations and mutations that cause allelic loss. 
     
     
       7. The method of  claim 1  wherein said about 50% decrease from the normal level of full-length BRCA1 or full-length BRCA2 is the result of a mutation selected from the group consisting of nonsense mutations, frameshift mutations, promoter mutations, enhancer mutations, splice site mutations, null mutations, and poly-A tail mutations. 
     
     
       8. The method of  claim 1  wherein said biological sample is selected from the group consisting of body fluids containing cells and tissue specimens. 
     
     
       9. The method of  claim 8  wherein said body fluids are selected from the group consisting of blood, serum, plasma, semen, breast exudate, gastric secretions, fecal suspensions, bile, saliva, tears, sputum, mucous, urine, lymph, cytosols, ascites, pleural effusions, amniotic fluid, bladder washes, bronchoalveolar lavages, and cerebrospinal fluid. 
     
     
       10. The method of  claim 1  wherein said normal cells are peripheral blood lymphocytes. 
     
     
       11. The method of  claim 1  wherein said method is diagnostic for hereditary breast cancer or a predisposition to hereditary breast cancer, or is diagnostic/prognostic for hereditary breast cancer. 
     
     
       12. The method of  claim 1  wherein the levels of full-length BRCA1 and full-length BRCA2 are determined by Western blot analysis, by immunoprecipitation and then by Western blot analysis, by flow cytometry, by EIA, by ELISA, by RIA, by competition immunoassay, by dual antibody sandwich assay, by chemiluminescent assay, by bioluminescent assay, by fluorescent assay, or by agglutination assay. 
     
     
       13. The method of  claim 1  which is automated.